Method for the production of pure virally inactivated butyrylcholinesterase

ABSTRACT

The present invention provides a method for purifying butyrylcholinesterase from various biological fluids. Biological fluids include, e.g., blood, blood fractions, plasma, and bioreactor broths, and other such mixtures containing butyrylcholinesterase. In one embodiment, the invention provides a method for the production of purified, virally inactivated butyrylcholinesterase by contacting a biological fluid containing butyrylcholinesterase with a cationic exchange chromatography material, with an affinity chromatography material, and treating the fluid with solvent detergent. The resulting purified butyrylcholinesterase can also be subjected to a pasteurization step, and formulated in a sodium chloride/sodium phosphate solution for storage or lyophilization.

FIELD OF THE INVENTION

[0001] The present invention provides a method for the isolation andpurification of butyrylcholinesterase from complex biological fluidssuch as plasma. More specifically, the present invention provides amethod for purifying and disinfecting butyrylcholinesterase frombutyrylcholinesterase-containing biological fluids, e.g., Cohn FractionIV-4 or IV-1, comprising subjecting such fluids to cationic exchangechromatography, affinity chromatography, a solvent detergent treatment,and pasteurization. As discussed more fully below, the order of thosesteps can be varied.

BACKGROUND OF THE INVENTION

[0002] Butyrylcholinesterase is an enzyme found mainly in plasma.Although the normal physiological role of butyrylcholinesterase isunknown, butyrylcholinesterase has been shown to metabolizeacetylcholine, degrade cocaine, and inactivate anaesthetic drugs andmuscle relaxants, including succinylcholine, succinylcholine-likecompounds and mivacurium (Gorelick et al., Drug Alcohol Depend., 48(3):159-65, 1997; Stewart et al., Clin. Pharmacol. Ther., 25: 464-8, 1979;Krasowski et al., Can. J. Anaesth., 44:525-34, 1997; Jatlow et al.,Anesth. Anag., 58:235-8, 1979). Butyrylcholinesterase has also beenshown to act as an antidote to nerve gas and organo-phosphorus compounds(Ashani et al., Biochem. Pharmacol., 41:37-41, 1991; Broomfield et al.,JPET, 259:633-8, 1991; Doctor et al., New Approaches to MedicalProtection Against Chemical Warfare Nerve Agents In Chemical WarfareAgents: Toxicity at Low Levels, pp. 191-214, Lewis Publishers, Inc.,2001; Ashani et al., Drug Development Research, 50:298-308, 2000).

[0003] Previous methods for isolating butyrylcholinesterase involvedeither ammonium sulfate precipitations or electrophoresis, whichresulted in yields of butyrylcholinesterase of about 10% and purities of10% or less (Goedde et al., Humangenetik., 1:311-8 1965; Haupt et al.,Blut., 14(2):65-75, 1966). Present methods employing chromatography ofplasma result in yields of butyrylcholinesterase ranging from 30-40% to63% (Grunwald et al., J. Biochem. Biophys. Methods, 34(2):123-35, 1997;Lockridge et al., J. Biol. Chem., 287:12012-8, 1982). In order toevaluate butyrylcholinesterase for its therapeutic and pharmacologicalproperties, large quantities of purified, virally inactivatedbutyrylcholinesterase are needed.

[0004] Within the art, there remains a need for a method whereby largequantities of virally inactivated butyrylcholinesterase can be isolatedfrom biological fluids such as plasma in a highly purified form. Untilnow, there have been no commercially viable, easily performed methods toefficiently and economically produce large quantities of purified,virally inactivated butyrylcholinesterase from biological fluids. Themethods of the present invention address that need.

SUMMARY OF THE INVENTION

[0005] To satisfy the need in the art, we have developed a versatile,commercially viable method for producing purified, virally inactivatedbutyrylcholinesterase at high yields and purity. Specifically, thepresent invention provides a novel method for recovering purified,virally inactivated butyrylcholinesterase from a biological fluid suchas plasma, Cohn plasma Fraction IV-4 or IV-1, analogous blood fractions,or fluids from bioreactors. The various embodiments of the methodcomprise subjecting a butyrylcholinesterase-containing fluid to a seriesof steps including cationic exchange chromatography, affinitychromatography, solvent detergent treatment, and pasteurization. Thosesteps need not be performed in that order, and the methods can besuccessfully performed with duplication of one or more of those steps.

DETAILED DESCRIPTION OF THE INVENTION

[0006] The inefficient and complex aspects of butyrylcholinesterasepurification and viral inactivation can be eliminated using a simpletechnique employing one or more steps incorporating cation exchangechromatography and affinity chromatography. The method of the inventioncan be used to produce purified and virally inactivatedbutyrylcholinesterase from plasma or plasma fractions in yields rangingfrom about 70% to about 80%. The term pure or purified is used herein torefer to a purity of greater than about 70%. Preferred embodimentsafford butyrylcholinesterase in purity of about 80% or greater. The morepreferred embodiments afford butyrylcholinesterase in purity of about90% or greater.

[0007] The purity of butyrylcholinesterase is determined by SDS-PAGEanalysis and by the specific activity of the final product. Enzymaticactivity of the purified butyrylcholinesterase is expressed as mg/mg orunits/mg.

[0008] Butyrylcholinesterase can be isolated frombutyrylcholinesterase-containing sources such as commercially availableplasma, plasma fractions such as Cohn Fraction IV-1 or Cohn FractionIV-4, a mixed plasma fraction of Cohn Fraction IV-1 and IV-4,recombinant sources, and other biological samples. If plasma is used,the plasma can be treated to produce Cohn Fraction IV-4 or IV-1, orplasma fractions of similar composition, as set forth in Cohn et al. (J.Amer. Chem. Soc., 68:459, 1946), or by other methods known in the art.

[0009] In accordance with one embodiment of the invention, there isprovided a method for producing purified, virally inactivatedbutyrylcholinesterase from human plasma, in particular Cohn plasmaFraction IV-4. One preferred example of the method comprises subjectinga solution of Cohn plasma Fraction IV-4 to cation exchangechromatography followed by affinity chromatography. A solvent detergenttreatment step may be added to inactivate lipid-enveloped viruses; andone or more of the chromatographic steps can be duplicated. Thepurification portion of the method is versatile, and the order ofperforming the various steps is not crucial. The purifiedbutyrylcholinesterase is then pasteurized as a general pathogeninactivation step and to inactivate viruses.

[0010] The cation exchange chromatography step is performed using anyone of a wide variety of cation exchange materials, for example cationexchangers linked to supports such as agarose, dextran, cellulose,polyacrylamide, polystyrene, acrylic polymers, vinyl polymers, andsilica. Cation exchangers such as carboxymethyl and sulfopropyl moietiescan be linked to, e.g., agarose to produce carboxymethyl-agarose (e.g.,CM-SEPHAROSE®) and sulfopropyl-agarose (e.g., SP-SEPHAROSE®),respectively. CM-SEPHAROSE® is a preferred cation exchange material inmethods of the present invention.

[0011] Cation exchange chromatography can be performed by any of theknown methods in the art. Those skilled in the art will appreciate thatany conventional format for effectively exploiting cation exchangechromatography materials will be suitable. In a preferred embodiment,column chromatography is used. In such an embodiment, the cationexchange chromatography column is packed with CM-SEPHAROSE® andequilibrated with buffer, preferably about 25 mM sodium acetate, to a pHranging from about 4.8 to about 5.2 and a conductivity ranging fromabout 0.85 to about 6.0 mS. Other equilibration buffers encompassed bythe method of the invention include but are not limited to sodiumcitrate and sodium phosphate.

[0012] The fall through from the cation ion exchange column contains thebutyrylcholinesterase. The resulting biological fluid is adjusted to pHof about 6.0 to about 8.5, more preferably to a pH of about 7.5. Thefall through is then concentrated about 5 to about 10 fold by methodsknown in the art, for example by ultrafiltration or diafiltration, andadjusted to a pH ranging from about 6.0 to about 8.5 and a conductivityranging from about 6.0 to about 15 mS.

[0013] At this stage of the butyrylcholinesterase purificationprocedure, the fall through concentrate can be solvent-detergent treatedbefore or after an affinity chromatography step. Solvent-detergenttreatment involves mixing the fall through or eluate with a solvent suchas Tri-n-Butyl Phosphate (TnBP) mixed with a detergent such as thenon-ionic detergent Tween-80 or Triton X-100, or combinations thereof,at a concentration ranging from about 0.3% to about 1.0% (w/v) for eachsolvent-detergent used. Solvent-detergent treatment of the eluate afteraffinity chromatography is preferable because the volume of eluate beingtreated is much smaller.

[0014] The fall through concentrate from the cation exchangechromatography step is then subjected to affinity chromatography. Again,any conventional method for performing a purification step usingaffinity chromatography material is acceptable; however, the preferredmethod is column chromatography. Affinity chromatography materialsinclude, but are not limited to, amino acid resins, peptide ligandresins, antibody resins, carbohydrate resins, avidin/biotin resins, dyeresins, glutathione resins, hydrophobic resins, immunochemical resins,lectin resins, nucleic acid resins and nucleotide/coenzyme resins. Theterm “peptide ligand resins” is used to refer to affinity chromatographymatrices to which peptides (i.e., chains of two or more amino acids) arecoupled.

[0015] In a preferred embodiment of the invention, affinitychromatography is performed using any one of a wide variety of affinitychromatography ligands linked to conventional supports. Conventionalsupports are matrices such as gels or resins and include, but are notlimited to, agarose, dextran, cellulose, polystyrene, acrylic resins,acrylamides, vinyl resins, and cross-linked and/or derivatizedvariations thereof. One of skill in the art will appreciate that theaffinity chromatography support material can be chosen from a variety ofcommercially available materials, and will be selected from among thevarious materials such that it has the requisite pore size toaccommodate butyrylcholinesterase.

[0016] A preferred affinity chromatography material is that in which thesupport is agarose covalently binding procainamide. Another preferredaffinity chromatography material is an agarose support covalentlybinding a butyrylcholinesterase-binding peptide. Examples of suchpeptides are presented below.

[0017] Commercially available examples of suitable supports includeSEPHAROSE® and SEPHADEX® affinity chromatography resins. One of skill inthe art will appreciate that many of the supports that are commonlyemployed in affinity chromatography materials, and which have anappropriate pore size for butyrylcholinesterase will be useful in thepresent invention. Those supports may be purchased, and preferred ligandcan be subsequently coupled to the resin.

[0018] As stated above, various materials can be used to performaffinity purification of butyrylcholinesterase from biological fluids.For example, procainamide affinity chromatography material can beemployed. Another option is a chromatography material to which is boundan antibody that binds butyrylcholinesterase. Alternatively, otherproteins or compounds such as peptide ligands or inhibitors that bindbutyrylcholinesterase can be coupled to the resin. Methods for usingthose additional materials (i.e., antibodies, substrates, compounds,inhibitors, etc.) for affinity chromatography are known within the art.

[0019] In one embodiment, the present invention provides a method forproducing purified, virally inactivated butyrylcholinesterasecomprising: contacting a butyrylcholinesterase-containing biologicalfluid with a cation exchange chromatography material; solvent-detergenttreating the biological fluid; contacting the biological fluid with abutyrylcholinesterase-binding affinity chromatography material;recovering bound butyrylcholinesterase from the affinity chromatographymaterial; and pasteurizing the recovered butyrylcholinesterase. Althoughit will be appreciated that certain steps will be performedsequentially, it is an advantage of the present invention that severalof the foregoing steps can be interchanged in order without sacrificingthe effectiveness of the method. Moreover, the purity of the recoveredbutyrylcholinesterase can be improved by repeating one or more of thesteps, particularly the affinity chromatography step.

[0020] The method affords the added versatility that when duplicatingsuch steps, different materials can be used. Thus, if one chooses torepeat the affinity chromatography step, different chromatography mediacan be employed in the two different steps.

[0021] Butyrylcholinesterase can be affinity purified frombutyrylcholinesterase containing sources using peptide ligands that bindbutyrylcholinesterase. In such an embodiment, peptide ligands that bindbutyrylcholinesterase are first identified. This can be done byscreening combinatorial peptide libraries using labeledbutyrylcholinesterase. Butyrylcholinesterase can be labeled using any ofthe conventional methods in the art, such as radiolabeling. Peptideligands that are positive for binding butyrylcholinesterase can bedetected by using known methods in the art. For example, a conventionalenzyme activity assay using butyrylcholine and DTNB(5,5-Dithiobis-2-Nitrobenzoic Acid) can be used. The peptide ligandsfound to bind butyrylcholinesterase can then be sequenced, andreproduced for isolating and purifying butyrylcholinesterase.

[0022] The positive peptide ligands can then be used in such anisolation and purification process in otherwise conventional affinitychromatography methods. In one embodiment, the ligands identified ashaving the required affinity for butyrylcholinesterase are immobilizedon an affinity chromatography support or matrix material. Any commonaffinity chromatography support or matrix material with the requisitepore size will likely be suitable.

[0023] In a preferred embodiment, the ligand(s) are covalently attachedto an affinity chromatography matrix to create a matrix-ligandcomposite. A biological fluid containing butyrylcholinesterase iscontacted with the matrix-ligand composite, and butyrylcholinesterase isconcentrated on the composite. Extraneous matter and undesiredcomponents from the biological fluid are washed away; and thebutyrylcholinesterase is subsequently eluted from the composite withappropriate buffers and solutions, and the butyrylcholinesterase isrecovered.

[0024] The term “biological fluid” is used herein to refer to an aqueousfluid or mixture containing various biological constituents andcontaminants in combination with butyrylcholinesterase. In short, abiological fluid, as used herein, is an aqueous mixture of impurebutyrylcholinesterase. The butyrylcholinesterase in the biological fluidis either natural or produced from recombinant sources. Examples ofbiological fluids include blood; blood fractions, plasma, plasmafractions, extracts, and isolates; cell or tissue homogenates, extracts,or isolates; and bioreactor broths or other reaction mixtures suitablefor making and/or recovering butyrylcholinesterase. Preferred biologicalfluids for recovering natural butyrylcholinesterase are Cohn FractionsIV-4, Cohn Fraction IV-1, Precipitate IV (hereinafter “PPT. IV”; alsoreferred to in the art as Precipitate B or PPT. B) from theKistler-Nitschmann fractionation, and combinations thereof. For adescription of PPT. IV see, Kistler P. and Nitschmann H., Vox.Sanguinis, 7, 414 (1960).

[0025] As discussed below, the various biological fluids, such as plasmafractions, can be reconstituted in water or other appropriate aqueoussolvents to achieve the desired density, product concentration, and thelike. Selection of aqueous solvent and the amount used forreconstitution will vary depending upon the biological fluid employed,and can be routinely determined by one of ordinary skill in the art.

[0026] We have identified the following peptides as preferred peptideaffinity ligands for the concentration, separation, and purification ofbutyrylcholinesterase: AKDQIP (alanine, lysine, aspartic acid,glutamine, isoleucine, proline), AKGDQN (alanine, lysine, glycine,aspartic acid, glutamine, asparagine), WKDAVQ (tryptophan, lysine,aspartic acid, alanine, valine, glutamine), GFVGXA (glycine,phenylalanine, valine, glycine, X, alanine, wherein X is2-naphthylalanine), GFHGXI (glycine, phenylalanine, histidine, glycine,X, isoleucine, wherein X is 2-naphthylalanine), AFTNGE (alanine,phenylalanine, threonine, asparagine, glycine, glutamic acid), AFTNQE(alanine, phenylalanine, threonine, asparagine, glutamine, glutamicacid), GTNYHQ (glycine, threonine, asparagine, tyrosine, histidine,glutamine), AEVDPG (alanine, glutamic acid, valine, aspartic acid,proline, glycine).

[0027] In still another embodiment of the present invention,butyrylcholinesterase can be concentrated from a biological fluid in asimple separation of one or more steps. That is, the peptide ligandsidentified above can be used in a highly versatile and effectiveone-step method for concentrating and isolating butyrylcholinesterasefrom biological fluids. Thus, the present invention provides a methodfor concentrating butyrylcholinesterase comprising contacting abutyrylcholinesterase-containing biological fluid with a peptide ligandaffinity chromatography material wherein the peptide ligand is selectedfrom the group consisting of: a) alanine, lysine, aspartic acid,glutamine, isoleucine, and proline; b) alanine, lysine, glycine,aspartic acid, glutamine, and asparagine; c) tryptophan, lysine,aspartic acid, alanine, valine, and glutamine; d) glycine,phenylalanine, valine, glycine, 2-naphthylalanine, and alanine; e)glycine, phenylalanine, histidine, glycine, 2-naphthylalanine, andisoleucine; f) alanine, phenylalanine, threonine, asparagine, glycine,and glutamic acid; g) alanine, phenylalanine, threonine, asparagine,glutamine, and glutamic acid; h) glycine, threonine, asparagine,tyrosine, histidine, glutamine; and i) alanine, glutamic acid, valine,aspartic acid, proline, glycine; and recovering butyrylcholinesterasebound to said peptide ligand chromatography material. The contactingstep can be a standard column chromatography method; and the recoverycan be performed by eluting the butyrylcholinesterase from the columnusing known buffers, salt solutions, and solvents.

[0028] The simple one-step separation method can be combined with one ormore purification and/or disinfection steps. Thus, the peptide ligandaffinity separation step can be coupled with a cation exchange step, asolvent detergent treatment step, and/or a pasteurization step.

[0029] Further, one or more of the purification steps (i.e., affinity orcation exchange) can be duplicated in a variation of the method. Induplicating one or more of those steps, different media can be employed.For example, in duplicating the affinity separation step, it is possibleto use a different peptide ligand or a different class of ligand (e.g.,procainamide).

[0030] Butyrylcholinesterase can be affinity purified using a variety ofaffinity chromatography media. In another preferred embodiment,procainamide affinity reagent (p-amino-N-(2-diethylaminoethyl)benzamide) is covalently coupled to 6-aminohexanoic acid agarose usingEDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) as a couplingreagent. The affinity chromatography column with procainamide affinityreagent is equilibrated with buffer, preferably 20 mM sodium phosphateand 0.1 M sodium chloride, to a pH ranging from about 6.0 to about 8.5and a conductivity ranging from about 8 to about 15 mS. Otherequilibration buffers encompassed by the method of the invention includebut are not limited to Tris and glycine buffers.

[0031] As stated above, the eluate from the affinity chromatographycolumn can be solvent-detergent treated. If solvent-detergent treatmentis performed at this stage of butyrylcholinesterase isolation, then thissolvent-detergent treated eluate is subjected to a second round ofaffinity chromatography, and the butyrylcholinesterase is eluted fromthe column with sodium chloride ranging from about 0.3 to about 0.5 M.Solvent-detergent treatment is performed as described above.

[0032] The methods of the present invention can be used to inactivatelipid enveloped or non-lipid enveloped viruses. Therefore, the purifiedbutyrylcholinesterase is pasteurized for a time period ranging fromabout 8 to about 72 hours, preferably from about 8 to about 24 hours,without substantial loss of butyrylcholinesterase activity.

[0033] The present methods afford viral inactivation with retention ofat least about 80% butyrylcholinesterase activity even afterpasteurization for about 72 hours. In one embodiment, pasteurization isperformed at about +60° C. for about 8 to about 12 hours in solutions ofL-lysine (about 0.1 to about 1.0 M) and sodium citrate (about 0.5 toabout 1.0 M); sucrose (about 0.3 to about 0.6 M) and sodium citrate(about 0.6 to about 1.0 M); or sucrose (about 0.6 to about 1.0 M) andglycine (about 0.3 to about 0.5 M). Preferably, pasteurization isperformed in a solution of sucrose (about 0.3 to about 0.6 M) and sodiumcitrate (about 0.6 to about 1.0 M).

[0034] Esterolytic activity of butyrylcholinesterase was determined in astandard esterolytic assay with the substrate butyrylthiocholine.Retention of esterolytic activity of butyrylcholinesterase for about 24hours at about +60 C was achieved in formulations of about 0.3 to about0.6M sucrose in varying combinations with about 0.6 to about 1.0 Msodium citrate as well as in a formulation of about 0.1 M lysine andabout 1.0 M sodium citrate, as preferred pasteurization formulations.

[0035] The following Examples are provided to further illustrate a fewembodiments of the present invention. The Examples are presented forillustration only, and do not reflect or suggest any limitation on thescope of the invention.

EXAMPLE I Purification and Viral Inactivation of Butyrylcholinesterase

[0036] Materials: Cohn Fraction IV-4 paste obtained by the Cohn coldethanol fractionation process of pooled human plasma was used in thisexample. The cation exchange materials used (CM-SEPHAROSE®, FAST FLOW®OR SP-SEPHAROSE®, FAST FLOW®) are commercially available resins.

[0037] The affinity resin was prepared by coupling procainamidecovalently to ECH-SEPHAROSE 4B® (Pharmacia) using EDC as a couplingreagent. Procainamide was added in 5-fold molar excess per gram ofswollen gel relative to the resin ligands. EDC was then added to a finalconcentration of about 0.1 M. The coupling procedure was performed indistilled water, adjusted to a pH ranging from about 4.5 to about 5.5with HCl. The procainamide/EDC mixture was rotated gently for about 24hours at room temperature, and the resin was subsequently washed withseveral cycles of high and low pH solutions (0.1 M acetate buffer, 0.5 MNaCl pH 4.0; 0.1 M Tris-HCl buffer; 0.5 M NaCl, pH 8.0), followed bywashes with distilled water. The resulting resin was stored in about 20%ethanol.

[0038] In this example, the method of the invention was performed asfollows: 1.0 kg of Cohn IV-4 was suspended in about 8.0 L of distilledwater (optionally containing 1.0 mM EDTA) to form a mixture and stirredfor about 2 hours or overnight at about 4° C. Particulate matter such asinsoluble proteins and Celite, an additive added to collect CohnFraction IV-4, was removed by centrifugation at about 4000 g andclarified through 5 μm, 1.0 μm, 0.5 μm and 0.2 μm filters. Afteradjusting the pH to a range from about 4.9 to about 5.1 with acetic acidand the conductivity to between about 0.85 to about 1.3 mS withdistilled water, the mixture was loaded on a cation exchangechromatography column packed with approximately 0.35 L-0.5 L ofCM-SEPHAROSE®, which had been equilibrated with about 25 mM sodiumacetate (pH 4.9; conductivity 1.8 mS; temperature 22° C.), at a linearvelocity of about 60 cm/H and a residence time ranging from about 10 toabout 12 minutes (if SP-SEPHAROSE® resin is used, the pH is about 5.2and the conductivity is about 6.0 mS). The column was washed with about0.5 L of 25 mM sodium acetate buffer (pH 4.9), and the wash was added tothe fall through to collect the butyrylcholinesterase quantitatively.The specific advantage of this chromatographic step was that thebutyrylcholinesterase remained in the fall through while greater than90% of the contaminating proteins were bound to the cation exchangematerial.

[0039] The fall through, which contains the butyrylcholinesterase, wasadjusted to a pH of about 7.5 with about 1.0 M NaOH and thenconcentrated 5 to 10 fold by ultrafiltration using a membrane with amolecular weight cutoff of about 100,000. After concentrating the fallthrough, the conductivity was adjusted to about 9.5 mS with about 4.0 MNaCl.

[0040] The concentrate from above was then loaded onto an affinitychromatography column packed with approximately 0.05-0.075 L ofprocainamide (PAM) affinity resin, which had been equilibrated withabout 20 mM sodium phosphate and about 0.1 M sodium chloride (pH 7.5;conductivity 10.5 mS), at a linear velocity of about 50 cm/H. The columnwas washed step-wise with about 0.1, 0.15 , 0.175 and 0.2 M NaCl inabout 20 M sodium phosphate (pH 7.5) and then eluted with about 0.4 L of0.5 M NaCl and 20 mM sodium phosphate (pH 7.5).

[0041] At this stage of the butyrylcholinesterase purificationprocedure, the eluted butyrylcholinesterase was treated withsolvent-detergent. Solvent-detergent treatment involved mixing the PAMaffinity eluate fall through with about 1% (v/v) Tween-80 and about 0.3%(v/v) TNBP.

[0042] The eluate was then subjected to a second round of PAM affinitychromatography and washed and eluted as described above. The yield andpurity of the isolated butyrylcholinesterase was calculated to be about80% and about 80-90%, respectively.

[0043] To inactivate both lipid and non-lipid enveloped viruses, such asporcine parvo virus (PPV) (which is used as a model virus for human B19parvovirus), in the purified butyrylcholinesterase, the eluate from thesecond PAM affinity chromatography step was pasteurized at about +60° C.for about 24 hours in a solution of sucrose and sodium citrate or lysineand sodium citrate or glycine and sucrose.

[0044] We found that combinations of sucrose (about 0.3 to about 0.6 M)with varying concentrations of sodium citrate (about 0.6 to about 1.0 M)or the combination of lysine (about 0.1 to about 0.5 M) with sodiumcitrate (about 1.0 M) allowed pasteurization of butyrylcholinesterase inliquid form at about +60° C. with greater than 95% butyrylcholinesteraseactivity remaining and greater than a 10⁴ reduction in PPV. The finalbutyrylcholinesterase product can be subjected to diafiltration orultrafiltration before formulation. Butyrylcholinesterase can beformulated in liquid form in about 0.1 to about 0.15 M NaCl and about0.02 M sodium phosphate buffer (pH about 6.5 to about 7.5) and stored inliquid for or it can be lyophilized.

[0045] The process may readily be scaled up. For example, whenprocessing about 150 kg of biological fluid, such as Cohn Fraction IV-1or IV-4, we recommend using approximately 55-70 L of cation exchangeresin and approximately 7.5-10 L of PAM resin.

EXAMPLE II Affinity Purification of butyrylcholinesterase Using PeptideLigands

[0046] Identification of positive peptides: Solid phase combinatorialpeptide libraries were synthesized on polymethacrylate beads (Buettneret al, Int. J. Pep Prot. Res., 47, 70-83 (1996)) and screened for thebinding of butyrylcholinesterase using the radiolabeled technique ofJentoft et al. (Methods in Enzymology, 91:570-79 (1983)) to radiolabelbutyrylcholinesterase. To detect positive ligands that bindbutyrylcholinesterase, the screening method of Mondorf et al. (J.Peptide Res., 52(6):526-36 (1998)) combined with an activity assay usingthe substrate butyrylthiocholine and DTNB (5,5-Dithiobis-2-NitrobenzoicAcid) were used. This stained the beads that bound activebutyrylcholinesterase yellow.

[0047] We have identified the following ligands as useful and effectivefor the affinity purification of butyrylcholinesterase: AKDQIP (alanine,lysine, aspartic acid, glutamine, isoleucine, proline), AKGDQN (alanine,lysine, glycine, aspartic acid, glutamine, asparagine), WKDAVQ(tryptophan, lysine, aspartic acid, alanine, valine, glutamine), GFVGXA(glycine, phenylalanine, valine, glycine, X, alanine, wherein X is2-naphthylalanine), GFHGXI (glycine, phenylalanine, histidine, glycine,X, isoleucine, wherein X is 2-naphthylalanine), AFTNGE (alanine,phenylalanine, threonine, asparagine, glycine, glutamic acid), AFTNQE(alanine, phenylalanine, threonine, asparagine, glutamine, glutamicacid), GTNYHQ (glycine, threonine, asparagine, tyrosine, histidine,glutamine), AEVDPG (alanine, glutamic acid, valine, aspartic acid,proline, glycine).

[0048] Any of those ligands, or other butyrylcholinesterase-bindingpeptides, can be immobilized on an affinity matrix material, and used inaffinity separations methods to concentrate, isolate, and recoverbutyrylcholinesterase from, e.g., plasma, Cohn Fract IV-1, Cohn FractionIV-4, and other biological fluids containing butyrylcholinesterase incombination with other proteins and extraneous biological matter. Itwill be appreciated by those skilled in the art that, in view of thepresent disclosure, one of skill in the art will be able to identifyadditional such peptides having the desired level of affinity, andreversible binding properties with butyrylcholinesterase. Accordingly,the foregoing peptide ligands are illustrative only and the recitationof those ligands herein should not be construed as a limitation on thescope of the invention or of the potential ligands useful in the presentinvention.

[0049] Numerous modifications and variations of the methods of thepresent invention are possible in light of the foregoing disclosure.Therefore, one of skill in the art will understand that the inventioncan be exploited otherwise than as specifically described herein.

[0050] All references cited herein are incorporated herein by referencein their entirety.

1. A method for producing purified, virally inactivatedbutyrylcholinesterase comprising: a. contacting abutyrylcholinesterase-containing biological fluid with a cation exchangechromatography material; b. solvent-detergent treating the biologicalfluid; c. contacting the biological fluid with abutyrylcholinesterase-binding affinity chromatography material; d.recovering bound butyrylcholinesterase from the affinity chromatographymaterial; and e. pasteurizing the recovered butyrylcholinesterase. 2.The method of claim 1, wherein steps a, b, c, and d are performedsequentially or randomly, but where step d always follows step c.
 3. Themethod of claim 1, wherein either or both of steps a and c are repeated.4. The method of claim 1, wherein the butyrylcholinesterase containingbiological fluid comprises a plasma fraction selected from the groupconsisting of Cohn Fraction IV-4, Cohn Fraction IV-1, PPT. IV from aKistler-Nitschmann fractionation, and combinations thereof.
 5. Themethod of claim 1, wherein the butyrylcholinesterase-containingbiological fluid comprises recombinantly produced butyrylcholinesterase.6. The method of claim 1, wherein said cation exchange material is acation exchanger linked to a support selected from the group consistingof agarose, dextran, cellulose, polyacrylamide, polystyrene, acrylicpolymers, vinyl polymers, and silica.
 7. The method of claim 1, whereinsaid cation exchange material comprises a carboxymethyl moiety or asulfopropyl moiety.
 8. The method of claim 1, wherein said affinitychromatography material is selected from the group consisting of peptideligand resins, carbohydrate resins, dye resins, immunochemical resins,lectin resins, nucleic acid resins, and nucleotide/coenzyme resins. 9.The method of claim 1, wherein said affinity chromatography materialcomprises a support selected from the group consisting of agarose,dextran, cellulose, polystyrene, an acrylic resin, an acrylamide resin,and a vinyl resin, and covalently attached to said support isprocainamide.
 10. The method of claim 1, wherein said affinitychromatography material comprises an agarose support to which iscovalently bound procainamide or a butyrylcholinesterase-binding peptideligand.
 11. The method of claim 10, wherein thebutyrylcholinesterase-binding peptide ligand is selected from the groupconsisting of: a. alanine, lysine, aspartic acid, glutamine, isoleucine,and proline; b. alanine, lysine, glycine, aspartic acid, glutamine, andasparagine; c. tryptophan, lysine, aspartic acid, alanine, valine, andglutamine; d. glycine, phenylalanine, valine, glycine,2-naphthylalanine, and alanine; e. glycine, phenylalanine, histidine,glycine, 2-naphthylalanine, and isoleucine; f. alanine, phenylalanine,threonine, asparagine, glycine, and glutamic acid; g. alanine,phenylalanine, threonine, asparagine, glutamine, and glutamic acid; h.glycine, threonine, asparagine, tyrosine, histidine, glutamine; and i.alanine, glutamic acid, valine, aspartic acid, proline, glycine.
 12. Themethod of claim 1, wherein said pasteurization of butyrylcholinesteraseis performed at about +60° C. for a time period ranging from about 8 toabout 72 hours.
 13. The method of claim 1, wherein said pasteurizationof butyrylcholinesterase is performed at about +60° C. in aL-lysine/sodium citrate solution or a sucrose solution for a time periodof at least about 8 hours.
 14. The method of claim 13, wherein saidsucrose solution is Sucrose/Sodium Citrate or Sucrose/Glycine.
 15. Themethod of claim 1, wherein said pasteurized butyrylcholinesterase isformulated in about 0.1 to about 0.15 M NaCl and about 0.02 M sodiumphosphate.
 16. A method for producing purified, virally inactivatedbutyrylcholinesterase comprising: a. contacting abutyrylcholinesterase-containing biological fluid with a cation exchangechromatography material; b. contacting the biological fluid with abutyrylcholinesterase-binding affinity chromatography material; c.recovering butyrylcholinesterase from the affinity chromatographymaterial; d. solvent-detergent treating the butyrylcholinesteraserecovered from the affinity chromatography material; e. contacting thesolvent-detergent treated product with a butyrylcholinesterase-bindingaffinity chromatography material; f. recovering butyrylcholinesterasefrom the affinity chromatography material; and g. pasteurizing therecovered butyrylcholinesterase.
 17. The method of claim 16, wherein thebutyrylcholinesterase-containing biological fluid comprises a plasmafraction selected from the group consisting of Cohn Fraction IV-4, CohnFraction IV-1, PPT. IV from a Kistler-Nitschmann fractionation, andcombinations thereof.
 18. The method of claim 16, wherein thebutyrylcholinesterase-containing biological fluid comprisesrecombinantly produced butyrylcholinesterase.
 19. The method of claim16, wherein said cation exchange chromatography material comprises acarboxymethyl moiety or a sulfopropyl moiety.
 20. The method of claim16, wherein said affinity chromatography material is selected from thegroup consisting of peptide ligand resins, carbohydrate resins, dyeresins, immunochemical resins, lectin resins, nucleic acid resins, andnucleotide/coenzyme resins.
 21. The method of claim 16, wherein saidaffinity chromatography material comprises an agarose support to whichis covalently bound procainamide or a butyrylcholinesterase-bindingpeptide ligand.
 22. The method of claim 21, wherein thebutyrylcholinesterase-binding peptide ligand is selected from the groupconsisting of: a. alanine, lysine, aspartic acid, glutamine, isoleucine,and proline; b. alanine, lysine, glycine, aspartic acid, glutamine, andasparagine; c. tryptophan, lysine, aspartic acid, alanine, valine, andglutamine; d. glycine, phenylalanine, valine, glycine,2-naphthylalanine, and alanine; e. glycine, phenylalanine, histidine,glycine, 2-naphthylalanine, and isoleucine; f. alanine, phenylalanine,threonine, asparagine, glycine, and glutamic acid; g. alanine,phenylalanine, threonine, asparagine, glutamine, and glutamic acid; h.glycine, threonine, asparagine, tyrosine, histidine, glutamine; and i.alanine, glutamic acid, valine, aspartic acid, proline, glycine.
 23. Themethod of claim 16, wherein said pasteurization of butyrylcholinesteraseis performed at about +60° C. for a time period ranging from about 8 toabout 72 hours.
 24. The method of claim 16, wherein said pasteurizationof butyrylcholinesterase is performed at about +60° C. in aL-lysine/sodium citrate solution or a sucrose solution for at leastabout 8 hours.
 25. The method of claim 24, wherein said sucrose solutionis Sucrose/Sodium Citrate or Sucrose/Glycine.
 26. The method of claim16, wherein said pasteurized butyrylcholinesterase is formulated inabout 0.1 to about 0.15 M NaCl and about 0.02 M sodium phosphate.
 27. Amethod for producing purified, virally inactivated butyrylcholinesterasecomprising: a. contacting a butyrylcholinesterase-containing biologicalfluid with a carboxymethyl-agarose cation exchange chromatographymaterial; b. concentrating the fall through from the cation exchangechromatography step by ultrafiltration; c. contacting theultrafiltration concentrate with a procainamide affinity chromatographymaterial to bind butyrylcholinesterase; d. eluting butyrylcholinesterasefrom the procainamide affinity chromatography material; e.solvent-detergent treating the eluate from the procainamide affinitychromatography material with about 1% (v/v) Tween-80 and about 0.3%(v/v) TnBP; f. contacting the solvent-detergent treated eluate with aprocainamide affinity chromatography material to bindbutyrylcholinesterase; g. eluting butyrylcholinesterase from theprocainamide affinity chromatography material; and h. pasteurizing theeluted butyrylcholinesterase at about +60° C. for at least about 8hours.
 28. The method of claim 27, wherein the butyrylcholinesterasecontaining biological fluid comprises a plasma fraction selected fromthe group consisting of Cohn Fraction IV-4, Cohn Fraction IV-1, PPT. IVfrom a Kistler-Nitschmann fractionation, and combinations thereof. 29.The method of claim 27, wherein the butyrylcholinesterase containingbiological fluid comprises recombinantly produced butyrylcholinesterase.30. A method for producing purified, virally inactivatedbutyrylcholinesterase comprising: a. contacting abutyrylcholinesterase-containing biological fluid with acarboxymethyl-agarose cation exchange chromatography material; b.solvent-detergent treating the biological fluid with about 1% (v/v)Tween-80 and about 0.3% (v/v) TnBP; c. contacting the solvent-detergenttreated fall through with a procainamide affinity chromatographymaterial to bind butyrylcholinesterase; d. recoveringbutyrylcholinesterase from the procainamide affinity chromatographymaterial; and e. pasteurizing the recovered butyrylcholinesterase atabout +60° C. for at least about 8 hours.
 31. The method of claim 30,wherein the butyrylcholinesterase containing biological fluid comprisesa plasma fraction selected from the group consisting of Cohn FractionIV-4, Cohn Fraction IV-1, PPT. IV from a Kistler-Nitschmannfractionation, and combinations thereof.
 32. The method of claim 30,wherein the butyrylcholinesterase-containing biological fluid comprisesrecombinantly produced butyrylcholinesterase.
 33. A method for producingpurified, virally inactivated butyrylcholinesterase comprising: a.contacting a butyrylcholinesterase-containing biological fluid with acation exchange chromatography material; b. subjecting the biologicalfluid to affinity purification using a peptide ligand affinitychromatography material that binds butyrylcholinesterase; c. recoveringbutyrylcholinesterase from the peptide ligand affinity chromatographymaterial; d. solvent-detergent treating the biological fluid; e.contacting the solvent-detergent treated biological fluid with a peptideligand affinity chromatography material; f. recoveringbutyrylcholinesterase from the affinity chromatography material; and g.pasteurizing the recovered butyrylcholinesterase.
 34. The method ofclaim 33, wherein said butyrylcholinesterase containing biological fluidcomprises a plasma fraction selected from the group consisting of CohnFraction IV-4, Cohn Fraction IV-1, PPT. IV from a Kistler-Nitschmannfractionation, and combinations thereof.
 35. The method of claim 33,wherein said butyrylcholinesterase-containing biological fluid comprisesrecombinantly produced butyrylcholinesterase.
 36. The method of claim33, wherein said pasteurized butyrylcholinesterase is formulated inabout 0.1 to about 0.15 M NaCl and about 0.02 M sodium phosphate. 37.The method of claim 33, wherein said peptide ligand is selected from thegroup consisting of: a. alanine, lysine, aspartic acid, glutamine,isoleucine, and proline; b. alanine, lysine, glycine, aspartic acid,glutamine, and asparagine; c. tryptophan, lysine, aspartic acid,alanine, valine, and glutamine; d. glycine, phenylalanine, valine,glycine, 2-naphthylalanine, and alanine; e. glycine, phenylalanine,histidine, glycine, 2-naphthylalanine, and isoleucine; f. alanine,phenylalanine, threonine, asparagine, glycine, and glutamic acid; g.alanine, phenylalanine, threonine, asparagine, glutamine, and glutamicacid; h. glycine, threonine, asparagine, tyrosine, histidine, glutamine;and i. alanine, glutamic acid, valine, aspartic acid, proline, glycine.38. A method for producing purified, virally inactivatedbutyrylcholinesterase comprising: a. contacting abutyrylcholinesterase-containing biological fluid with a cation exchangechromatography material; b. solvent-detergent treating the biologicalfluid; c. contacting the biological fluid with at least one peptideligand affinity chromatography material that bindsbutyrylcholinesterase; d. recovering butyrylcholinesterase from thepeptide ligand affinity chromatography material; and e. pasteurizing theeluted butyrylcholinesterase.
 39. The method of claim 38, wherein saidbutyrylcholinesterase containing biological fluid comprises a plasmafraction selected from the group consisting of Cohn Fraction IV-4, CohnFraction IV-1, PPT. IV from a Kistler-Nitschmann fractionation, andcombinations thereof.
 40. The method of claim 38, wherein saidbutyrylcholinesterase-containing biological fluid comprisesrecombinantly produced butyrylcholinesterase.
 41. The method of claim38, wherein said pasteurized butyrylcholinesterase is formulated inabout 0.1 to about 0.15 M NaCl and about 0.02 M sodium phosphate. 42.The method of claim 38, wherein said at least one peptide ligand is apeptide selected from the group consisting of: a. alanine, lysine,aspartic acid, glutamine, isoleucine, and proline; b. alanine, lysine,glycine, aspartic acid, glutamine, and asparagine; c. tryptophan,lysine, aspartic acid, alanine, valine, and glutamine; d. glycine,phenylalanine, valine, glycine, 2-naphthylalanine, and alanine; e.glycine, phenylalanine, histidine, glycine, 2-naphthylalanine, and isoleucine; f. alanine, phenylalanine, threonine, asparagine, glycine, andglutamic acid; g. alanine, phenylalanine, threonine, asparagine,glutamine, and glutamic acid; h. glycine, threonine, asparagine,tyrosine, histidine, glutamine; and i. alanine, glutamic acid, valine,aspartic acid, proline, glycine.
 43. A method for isolatingbutyrylcholinesterase comprising contacting abutyrylcholinesterase-containing biological fluid with a peptide ligandaffinity chromatography material wherein the peptide ligand is selectedfrom the group consisting of: a. alanine, lysine, aspartic acid,glutamine, isoleucine, and pro line; b. alanine, lysine, glycine,aspartic acid, glutamine, and asparagine; c. tryptophan, lysine,aspartic acid, alanine, valine, and glutamine; d. glycine,phenylalanine, valine, glycine, 2-naphthylalanine, and alanine; e.glycine, phenylalanine, histidine, glycine, 2-naphthylalanine, andisoleucine; f. alanine, phenylalanine, threonine, asparagine, glycine,and glutamic acid; g. alanine, phenylalanine, threonine, asparagine,glutamine, and glutamic acid; h. glycine, threonine, asparagine,tyrosine, histidine, glutamine; and i. alanine, glutamic acid, valine,aspartic acid, proline, glycine; and recovering butyrylcholinesterasebound to said peptide ligand chromatography material.
 44. The method ofclaim 43, wherein the peptide ligand is covalently bound to a supportselected from the group consisting of agarose, dextran, cellulose,polyacrylamide, polystyrene, acrylic polymers, vinyl polymers, silica,and cross-linked versions thereof.
 45. The method of claim 43, furthercomprising contacting the biological fluid with a cation exchangechromatography material.
 46. The method of claim 43, further comprisingsubjecting the biological fluid to a solvent-detergent treatment step.47. The method of claim 43, further comprising subjecting the biologicalfluid to a pasteurization step.